Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis
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Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis. / Lonowski, Lindsey A; Narimatsu, Yoshiki; Riaz, Anjum; Delay, Catherine Marie Elander; Yang, Zhang; Niola, Francesco; Duda, Katarzyna; Ober, Elke A; Clausen, Henrik; Wandall, Hans H; Hansen, Steen H; Bennett, Eric P; Frödin, Morten.
In: Nature Protocols, Vol. 12, No. 3, 03.2017, p. 581-603.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis
AU - Lonowski, Lindsey A
AU - Narimatsu, Yoshiki
AU - Riaz, Anjum
AU - Delay, Catherine Marie Elander
AU - Yang, Zhang
AU - Niola, Francesco
AU - Duda, Katarzyna
AU - Ober, Elke A
AU - Clausen, Henrik
AU - Wandall, Hans H
AU - Hansen, Steen H
AU - Bennett, Eric P
AU - Frödin, Morten
PY - 2017/3
Y1 - 2017/3
N2 - This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.
AB - This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.
U2 - 10.1038/nprot.2016.165
DO - 10.1038/nprot.2016.165
M3 - Journal article
C2 - 28207001
VL - 12
SP - 581
EP - 603
JO - Nature Protocols
JF - Nature Protocols
SN - 1754-2189
IS - 3
ER -
ID: 173811979